U.S. patent application number 09/917964 was filed with the patent office on 2002-08-01 for methods for tissue culturing and transforming elite inbreds of zea mays l..
This patent application is currently assigned to Stine Biotechnology. Invention is credited to Held, Bruce Marvin, Wilson, Herbert Martin.
Application Number | 20020104131 09/917964 |
Document ID | / |
Family ID | 22754882 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020104131 |
Kind Code |
A1 |
Wilson, Herbert Martin ; et
al. |
August 1, 2002 |
Methods for tissue culturing and transforming elite inbreds of Zea
mays L.
Abstract
The present invention is directed to methods for the tissue
culture and transformation of elite inbreds of corn (Zea mays L.).
More specifically, the present invention is directed to a method
for initiating Type II callus from corn tissue. The present
invention is also directed to a method for enhancing the
intergration of foreign DNA in the transformation of corn using a
heat shock treatment. The present invention is further directed to
a method of transforming elite inbreds of corn using
Agrobacterium.
Inventors: |
Wilson, Herbert Martin;
(Ames, IA) ; Held, Bruce Marvin; (Ames,
IA) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
555 13TH STREET, N.W.
SUITE 701, EAST TOWER
WASHINGTON
DC
20004
US
|
Assignee: |
Stine Biotechnology
Ames
IA
|
Family ID: |
22754882 |
Appl. No.: |
09/917964 |
Filed: |
July 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09917964 |
Jul 30, 2001 |
|
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09203679 |
Dec 1, 1998 |
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Current U.S.
Class: |
800/320.1 ;
435/424 |
Current CPC
Class: |
A01H 4/008 20130101;
C12N 15/8205 20130101 |
Class at
Publication: |
800/320.1 ;
435/424 |
International
Class: |
C12N 015/82; C12N
005/04 |
Claims
What is claimed is:
1. A method for stimulating a high frequency production of Type II
callus from immature embryos of elite corn inbreds which comprises
culturing said embryos on a solid medium comprising sucrose and a
monosaccharide sugar, wherein the the concentration of said
monosaccharide sugar is between about 5 g/L and about 30 g/L.
2. The method of claim 1, wherein said monosaccharide sugar is
selected from the group consisting of glucose, maltose, lactose,
sorbitol and mannitol.
3. The method of claim 1, wherein said monosaccharide sugar is
glucose.
4. A method for transforming elite lines of corn using
Agrobacterium comprising the steps of: (a) co-cultivating an
immature embryo from said elite line with Agrobacterium capable of
transferring at least one gene to tissue of said elite line on a
solid medium to produce an infected embryo; (b) culturing the
infected embryo on a solid medium comprising an antibiotic; (c)
culturing the resulting tissue on a solid medium comprising a
selective agent to select for transformed tissue; (d) selecting
transformed tissue with growing Type II callus capable of forming
water tower embryo structures; and (e) regenerating plants from
said embryo structures.
5. The method of claim 4, wherein, said Agrobacterium is selected
from Agrobacterium one to two days after rescue from frozen
glycerol stocks.
6. The method of claim 4, wherein said co-culitvating is performed
at a temperature of 15.degree. C. to about 28.degree. C.
7. The method of claim 6, wherein said temperature is 19.degree.
C.
8. The method of claim 4, wherein a heat shock treatment is applied
during co-cultivation, said heat shock treatment comprising a
temperature of 35.degree. C. to 55.degree. C. for 10 minutes to 180
minutes.
9. The method of claim 8, wherein said heat shock treatment
comprises a temperature of 45.degree. C. for 30 minutes to 60
minutes.
10. The method of claim 8, wherein said heat shock is performed at
24 hours to 72 hours after initiation of co-cultivation.
11. The method of claim 10, wherein said heat shock is performed at
48 hours to 54 hours after initiation of co-cultivation.
12. The method of claim 4, wherein said medium coprising an
antibiotic further comprises a monosaccharide sugar.
13. The method of claim 12, wherein said monosaccharide sugar is
selected from the group consisting of glucose, maltose, lactose,
sorbitol and mannitol.
14. The method of claim 13, wherein the concentration of said
monosaccharide sugar is from 5 g/L to 30 g/L.
15. The method of claim 4, wherein the concentration of antibiotic
in the medium of step (b) is from 15 mg/L to 75 mg/L.
16. The method of claim 15, wherein the concentration of said
antibiotic is 50 mg/L.
17. The method of claim 4, which further comprises the step of:
(b1) culturing the resulting tissue on a solid medium comprising an
antibiotic and a selective agent.
18. The method of claim 17, wherein the tissue is cultured on the
medium for two passages.
19. The method of claim 18, wherein the first passage is on said
solid medium comprising a low concentration of antibiotic and the
second passage is on said solid medium comprising a high
concentration of antibiotic.
20. The method of claim 19, wherein said low concentration of
antibiotic is from 15 mg/L to 75 mg/L and said high concentration
of antibiotic is from 150 mg/L to 350 mg/L.
21. The method of claim 20, wherein said low concentration of
antibiotic is 25 mg/L and said high concentration of antibiotic is
250 mg/L.
22. A method for transforming elite lines of corn using
Agrobacterium comprising the steps of: (a) co-cultivating an
immature embryo from said elite line with Agrobacterium capable of
transferring at least one gene to tissue of said elite line on a
solid medium to produce an infected embryo; (b) culturing the
infected embryo on a solid medium comprising an antibiotic and a
monosaccharide sugar in an amount of from 5 g/L to 30g/L; (c)
culturing the resulting tissue on a solid medium comprising an
antibiotic and a selective agent; (d) culturing the resulting
tissue on a solid medium comprising a selective agent to select for
transformed tissue; (e) selecting transformed tissue with growing
Type II callus capable of forming water tower embryo structures;
and (f) regenerating plants from said embryo structures.
23. The method of claim 22, wherein said monosaccharide sugar is
selected from the group consisting of glucose, maltose, lactose,
sorbitol and mannitol.
24. The method of claim 22, wherein, said Agrobacterium is selected
from Agrobacterium one to two days after rescue from frozen
glycerol stocks.
25. The method of claim 22, wherein co-cultivation is performed at
a temperature of 19.degree. C.
26. The method of claim 22, wherein a heat shock treatment is
applied during co-cultivation, said heat shock treatment comprising
a temperature of 35.degree. C. to 55.degree. C. for 30 minutes to
60 minutes.
27. The method of claim 25, wherein said heat shock is performed at
24 hours to 72 hours after initiation of co-cultivation.
28. The method of claim 22, wherein the concentration of antibiotic
in the medium of step (b) is from 15 mg/L to 75 mg/L.
29. The method of claim 17, wherein the tissue is cultured on the
solid medium in step (c) for two passages.
30. The method of claim 29, wherein the first passage is on said
solid medium comprising a low concentration of antibiotic and the
second passage is on said solid medium comprising a high
concentration of antibiotic.
31. The method of claim 30, wherein said low concentration of
antibiotic is from 15 mg/L to 75 mg/L and said high concentration
of antibiotic is from 150 mg/L to 350 mg/L.
32. A method for transforming elite lines of corn using
Agrobacterium comprising the steps of: (a) co-cultivating at a
temperature of 19.degree. C. an immature embryo from said elite
line with Agrobacterium capable of transferring at least one gene
to tissue of said elite line on a solid medium to produce an
infected embryo, said Agrobacterium is selected from Agrobacterium
one to two days after rescue from frozen glycerol stocks; (b)
culturing the infected embryo on a solid medium comprising an
antibiotic at a concentration of 15 mg/L to 75 mg/L and a
monosaccharide sugar selected from the group consisting of glucose,
maltose, lactose, sorbitol and mannitol in an amount of from 5 g/L
to 30 g/L; (c) culturing the resulting tissue on a solid medium
comprising an antibiotic and a selective agent; (d) culturing the
resulting tissue on a solid medium comprising a selective agent to
select for transformed tissue; (e) selecting transformed tissue
with growing Type II callus capable of forming water tower embryo
structures; and (f) regenerating plants from said embryo
structures.
33. The method of claim 32, wherein the tissue is cultured on the
solid medium in step (c) for two passages.
34. The method of claim 33, wherein the first passage is on said
solid medium comprising a concentration of antibiotic of from 25
mg/L to 60 mg/L and the second passage is on said solid medium
comprising a concentration of antibiotic of from 200 mg/L to 300
mg/L.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention is directed to methods for the tissue
culture and transformation of elite inbreds of corn (Zea mays L.).
More specifically, the present invention is directed to a method
for initiating Type II callus from corn tissue. The present
invention is also directed to a method for enhancing the
integration of foreign DNA in the transformation of corn using a
heat shock treatment. The present invention is further directed to
a method of transforming elite inbreds of corn using
Agrobacterium.
[0002] The publications and other materials used herein to
illuminate the background of the invention, and in particular,
cases to provide additional details respecting the practice, are
incorporated by reference, and for convenience are referenced in
the following text by author and date and are listed alphabetically
by author in the appended bibliography.
[0003] Cells which undergo rapid division and are totipotent are
generally regarded as highly suitable targets for introduction of
DNA as a first step in the generation of transgenic plants. In
corn, one prolific source of such cells is the so-called Type II
callus (Armstrong and Green, 1985). Initiation of this type of
callus has been achieved using immature embryos of certain
non-elite corn inbred lines, most notably A188. Hybrid embryos with
this inbred as a parent have also been used successfully. There
are, however, no reports of high frequency initiation of Type II
callus from immature embryos or other tissue of elite corn inbreds.
Therefore, it is desired to develop a method for culturing tissue
of elite corn germplasm which results in reproducible and high
frequency initiation of Type II callus from elite corn
germplasm.
[0004] Introduction of genes into corn can be accomplished in
several ways e.g. (a) particle bombardment of cultured cells
(Gordon-Kamm et al., 1990), immature embryos (Koziel et al., 1993),
meristems (Lowe et al., 1995), (b) electroporation of immature
embryos (D'Halluin et al., 1992), cultured cells (Laursen et al.,
1994), (c) electroporation and/or polyethylene glycol treatment of
protoplasts (Rhodes et al., 1988; Omirulleh et al., 1993), and (d)
co-cultivation with Agrobacterium tumefaciens (Ishida et al., 1996;
Hei and Komari, 1997; Zhao et al., 1998). Agrobacterium
tumefaciens-mediated DNA delivery has a number of advantages.
Firstly, the time taken to produce transgenic plants is short when
compared to other methods. Secondly, transgenes are generally
inserted as single copies, increasing the efficiency with which
usable breeding material can be produced. Thirdly, high
efficiencies of DNA delivery can be achieved. For breeding purposes
it would be ideal to introduce genes via Agrobacterium tumefaciens
directly into elite corn lines.
[0005] Following introduction of foreign DNA into target cells and
subsequent cell division, selection is applied to identify those
cells in which integration and expression of the foreign DNA is
occurring. Any procedure which could increase the frequency with
which foreign DNA integrates and expresses would greatly improve
transformation protocols. A procedure for increasing the efficiency
of integration of DNA into elite corn germplasm is described
herein.
[0006] Initial methods of Agrobacterium-mediated corn
transformation which were developed, while effective for some
germplasm, do not allow for efficient transformation of elite
lines. Hei et al. (European Published Patent Application No. 604
662 A1) discloses a method for transforming monocotyledons using
Agrobacterium. In this method, plant tissues were obtained from the
monocotyledon maize and the tissues were exposed to Agrobacterium
during the tissue differentiation process. Hei et al. disclose a
maize transformation protocol using maize calli. Saito et al.
(European Published Patent Application No. 672 752 A1) disclose a
method for transforming monocotyledons using the scutellum of
immature embryos. Ishida et al. (1996) also disclose a method
specific for transforming maize by exposing immature embryos to A.
tumefaciens. The methods were optimized for inbred A188 maize
lines. Transformation frequencies ranged from 12% to 30% at their
highest for immature embryos from A188 lines that were 1.0-1.2 mm
in length. Maize lines derived from crosses of A188 had
significantly lower transformation frequencies ranging from 0.4% to
about 5.3%. A188 is not generally considered a commercially useful
line and Ishida et al. (1996) failed to obtain recovery of stable
transformants in lines other than those containing A188.
[0007] In a subsequent method of Agrobacterium-mediated corn
transformation (Zhao et al., 1998), efficient transformation of
elite lines was possible using non-LS salt medium for the tissue
culture steps, including the steps of contacting and co-cultivating
immature embryos with Agrobacterium. The media used in this method
may be based on N6 or MS salts. This method achieves high
transformation frequency of hybrids between elite lines and A188
(e.g., a A188.times.B73 hybrid), a result which, although higher,
is similar to the the transformation frequency achieved in the
initial transformation procedures. Although the transformation
frequency of Pioneer elite inbreds (0.6-14.4%) was lower than that
achieved for the hybrids, this method did result in the
transformation of elite corn inbreds.
[0008] Thus, it is desired to develop methods which allow for the
more efficient transformation of elite lines, i.e., methods which
allow for the introduction of genes into elite corn lines at very
high efficiency using Agrobacterium tumefaciens-mediated DNA
delivery.
SUMMARY OF THE INVENTION
[0009] The present invention is directed to methods for the tissue
culture and transformation of elite inbreds of corn (Zea mays L.).
More specifically, the present invention is directed to a method
for initiating Type II callus from corn tissue. The present
invention is also directed to a method for enhancing the
integration of foreign DNA in the transformation of corn using a
heat shock treatment. The present invention is further directed to
a method of transforming elite inbreds of corn using
Agrobacterium.
[0010] In accordance with one embodiment of the present invention,
Type II callus is initiated from corn tissue, preferably immature
embryo, by adding a monosaccharide to the callus initiation medium.
The preferred mononsaccharide is glucose.
[0011] In accordance with a second embodiment of the present
invention, the integration of foreign DNA into corn tissue in
transformation of corn tissue is enhanced by application of a heat
shock treatment following contact of the foreign DNA with the corn
tissue. In a preferred embodiment, the heat shock is conducted at a
temperature of 45.degree. C. for about 30 to about 180 minutes,
preferably about 30 to about 60 minutes, more preferably about 30
minutes, at a time from about 24 to about 72 hours after the
contact of the DNA with the corn tissue, preferably from 48 to 54
hours.
[0012] In a third embodiment of the present invention, the
transformation of elite corn inbreds is achieved at a high
frequency by co-cultivating corn tissue with Agrobacterium
tumefaciens. In one particular embodiment, the transformation of
elite corn inbreds is enhanced by using Agrobacterium freshly grown
from glycerol cultures stored at about -86.degree. C. In a second
specific embodiment, the frequency of transformation is enhanced by
co-cultivating corn tissue and Agrobacterium at 19.degree. C. In a
third particular embodiment, the frequency of transformation is
enhanced by using lower levels of cefotaxime in the culturing
media. In a further embodiment, the frequency of transformation of
elite corn inbreds is enhanced using a combination of all of these
techniques.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 shows a comparison of the transient expression of the
gus gene dimer by the 35S meter following co-cultivation of
immature embryos with Agrobacterium at 19.degree. C. and 25.degree.
C.
[0014] FIG. 2 shows an actively growing Type II callus having
"water tower" embryo structures.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention is directed to methods for the tissue
culture and transformation of elite inbreds of corn (Zea mays L.).
More specifically, the present invention is directed to an improved
method for initiating Type II callus from corn tissue. In this
improved callus initiation method, the frequency of induction of
Type II callus is enhanced. An enhanced frequency of Type II callus
induction is achieved by including a monosaccharide, preferably
glucose, as more fully described below in the callus induction
medium.
[0016] The present invention is further directed to an improved
method for transforming elite inbreds of corn using Agrobacterium.
Improvements in the frequency of Agrobacterium-mediated corn
transformation is achieved by (a) enhancing the integration of
foreign DNA into corn tissue using a heat shock treatment, and/or
(b) cocultivating corn tissue and Agrobacterium at 19.degree. C.,
and/or (c) using Agrobacterium freshly grown from glycerol stocks
stored at about -86.degree. C., and/or (d) using a low level of
cefotaxime in the culturing medium. Each of these aspects is more
fully described below.
[0017] As will be discussed in more detail below, immature embryos
are isolated from maize, and the immature embryos are co-cultivated
with Agrobacterium, preferably on a solid medium. It has been found
that the frequency of transformation of inbred corn lines can be
enhanced during the co-cultivation step by using one or more
transformation frequency enhancement techniques. These
transformation frequency enhancement techniques include: (1)
co-cultivating the immature embryos and Agrobacterium at about
19.degree. C.; (2) using Agrobacterium which has been recently
recovered from frozen glycerol stocks; and (3) subjecting the
co-cultivated immature embryos and Agrobacterium to a heat shock.
With respect to recently recovered Agrobacterium, it has been
discovered that the use of Agrobacterium source cultures recovered
from frozen glycerol stocks stored at about -86.degree. C. and
cultured on YP medium for one to two days prior to use results in
an enhanced transformation frequency. For the heat shock treatment,
the co-cultivated immature embryo and Agrobacterium are subjected
to a temperature of about 35.degree. C. to about 55.degree. C.,
preferably about 40.degree. C. to about 50.degree. C., more
preferably about 45.degree. C. for a period of 10-180 minutes,
preferably 20-90 minutes, more preferably 30-60 minutes, and most
preferably 30 minutes. The co-cultivated immature embryo and
Agrobacterium are subjected to the heat shock after 24-72 hours,
preferably 24-60 hours, more preferably 48-54 hours of
co-cultivation.
[0018] The Type II callus is then regenerated into plants. "Water
tower" structures are generally in evidence as soon as callus is
initiated from immature embryos. The desired Type II callus is
cultured on solid medium to regenerate plants. The Type II callus
is then regenerated into plants. Tissue containing a high frequency
of "water tower" embryos structures is selected (see FIG. 2) from
the callus initiated from normal and "infected" immature embryos.
This tissue is desirable since it allows for ready regeneration of
plants. This desired Type II callus is cultured on solid medium to
regenerate plants.
[0019] Following the co-cultivation period, the "infected" immature
embryo is cultured, preferably on a solid medium, to initiate the
generation of Type II callus. The immature embryos are incubated in
the presence of at least one antibiotic known to inhibit the growth
of Agrobacterium without the addition of a selective agent for
plant transformants. It has been found that the frequency of
initiation of Type II callus can be enhanced by using a staged
exposure to the antibiotic. In accordance with this embodiment of
the invention, the infected embryo is first cultured on a medium
having a low concentration of antibiotic and then on a medium
having a high concentration of antibiotic. For example, if
cefotaxime is the antibiotic, the low concentration is about 15
mg/L to about 75 mg/L, preferably about 25 mg/L to about 60 mg/L,
more preferably 50 mg/L, and the high concentration is about 150
mg/L to about 350 mg/L, preferably about 200 mg/L to about 300
mg/L, more preferably about 250 mg/L. It is preferred that the
infected embryos are initially subjected to callus initiation
without selection. Selection is added as callus initiation and Type
II callus growth progresses. It has also been found that the
frequency of initiation of Type II callus can be enhanced by
including a monosaccharide, preferably glucose, in the initial
callus initiation medium. The amount of monosaccharide which is
included is about 5 g/L to about 30 g/L, preferably about 10 g/L to
about 20 g/L, more preferably about 10 g/L. It has been found that
the initiation of Type II callus is enhanced for normal immature
embryos, as well as "infected" immature embryos.
[0020] As a first step for practicing the present invention,
immature embryos are isolated from maize and exposed to
Agrobacterium. Immature embryos are an intact tissue that is
capable of cell division to give rise to callus cells that can then
differentiate to produce tissues and organs of a whole plant.
Immature embryos can be obtained from the fertilized reproductive
organs of a mature maize plant. Exemplary methods for isolating
immature embryos from maize are described by Green and Phillips
(1976). Maize immature embryos can be isolated from pollinated
plants, as another example, using the methods of Neuffer et al.
(1982). Another method is shown in Zhao et al. (1998). The immature
embryos are preferably used at approximately 8 days to 14 days
after pollination, and in a particularly preferred embodiment about
9 days to about 12 days after pollination when donor plants are
grown at around 25.degree. to 30.degree.. Preferably, the embryos
exposed to Agrobacterium range from about 0.8 to 2.0 mm and in a
particularly preferred embodiment about 1.0 mm to about 1.5 mm in
size. Immature embryos are preferably aseptically isolated from the
developing ear and placed in sterile medium for use.
[0021] The Agrobacterium used to transform the embryos is modified
to contain a gene of interest. Preferably the gene is incorporated
into a gene vector, to be delivered to the embryo. A variety of
Agrobacterium species are known and Agrobacterium species employed
for dicotyledon transformation can be used. A number of references
review Agrobacterium-mediated transformation in monocots and
dicots. These include, among others, Hooykaas (1989); Smith et al.
(1995); Chilton (1993); and Moloney et al. (1993).
[0022] Many Agrobacterium employed for the transformation of
dicotyledonous plant cells contain a vector having a DNA region
originating from the virulence (vir) region of the Ti plasmid. The
Ti plasmid originates from Agrobacterium tumefaciens. Nucleic acid
containing a gene encoding a polypeptide to be expressed in maize
can be inserted into this vector. Alternatively, the gene can be
contained in a separate plasmid which is then inserted into the Ti
plasmid in vivo, in Agrobacterium, by homologous recombination or
other equivalently resulting processes. A vector has also been
developed which contains a DNA region originating from the
virulence (vir) region of Ti plasmid pTiBo542 (Jin et al., 1987)
contained in a super-virulent Agrobacterium tumefaciens strain A281
showing extremely high transformation efficiency. The plasmid
containing the gene of interest was incorporated into the virulent
Agrobacterium tumefaciens strain A281 since strain A281 is known to
have a high transformation efficiency (Hood et al., 1984; Komari et
al., 1986). This type of vector is known in the art as a
"superbinary vector" (see European Patent Application 0
604662A1).
[0023] Superbinary vectors are preferred vectors for the
transformation methods of this invention. Exemplary superbinary
vectors useful for introducing nucleic acid encoding polypeptide
for expression in a maize plant via Agrobacterium-mediated
transformation methods include the superbinary pTOK162 (as
discussed in Japanese Laid-Open Patent Application No. 4-222527).
This vector includes regions that permit vector replication in both
E. coli and A. tumefaciens. The plasmid includes a T-DNA region,
characteristic of Ti plasmids. Nucleic acid containing a gene
encoding a polypeptide to be expressed in maize is inserted in the
T-DNA borders. Other superbinary vectors are known and these
vectors can similarly be incorporated into Agrobacterium (see e.g.,
Komari (1990) for pTOK23).
[0024] Examples of genes useful for expression in transformed plant
cells are known in the art. Exemplary genes include, but are not
limited to, Bt genes or patatin genes for insect resistance; the
Hm1 gene and chitinase genes for disease resistance; the pat, bar,
EPSP synthase gene or ALS genes for herbicide resistance; genes
encoding proteins with altered nutritional properties; genes
encoding enzymes involved in starch or oil biosynthetic pathways;
down-or up-regulatory sequences for metabolic pathway enzymes; and
the like. As those of ordinary skill in the art will recognize,
this is only a partial list of possible genes that can be used with
the transformation method of the present invention. Furthermore, as
those of ordinary skill in the art will also recognize, regulatory
sequences including promoters, terminators and the like will also
be required, and these are generally known in the art. Zhao et al.
(1998) discloses the construction of a prior art superbinary vector
pPHP 10525. This vector contains virB, virC and virG genes isolated
from superviral strain A281. The vector includes 35Sbar and ubi/GUS
plant expression cassettes inserted between the T-DNA borders.
Plant expression cassettes preferably comprise a structural gene to
which is attached regulatory DNA regions that permit expression of
the gene in plant cells. The regulatory regions consist at a
minimum of a promoter capable of directing expression of a gene in
a plant cell. The promoter is positioned upstream or at the 5' end
of the gene to be expressed. A terminator is also provided as a
regulatory region in the plant expression cassette and is capable
of providing polyadenylation and transcription terminator functions
in plant cells. The terminator is attached downstream or at the 3'
end of the gene to be expressed. Marker genes, included in the
vector, are useful for assessing transformation frequencies in this
invention.
[0025] The nucleic acid encoding a polypeptide for expression in
maize is inserted into the T-DNA region of the superbinary vector
using suitable restriction endonuclease recognition sites, by
homologous recombination, or the like. General molecular biological
techniques used in this invention are provided, for example, by
Sambrook et al. (1989) and the use of homologous recombination to
incorporate nucleic acid into plasmids contained in Agrobacterium
tumefaciens is disclosed by Herrera-Esterella et al. (1983) and
Horsch et al., (1984). The recombinant plasmid is selected in
Agrobacterium based on the use of a selectable marker incorporated
into the plasmid. Generally these markers are nucleic acid encoding
proteins that typically confer antibiotic resistance.
[0026] Plasmids are introduced into Agrobacterium using methods
known in the art, including the triple-cross method disclosed by
Ishida et al. (1996) or the method disclosed by Zhao et al.
(1998).
[0027] Agrobacterium containing the plasmid of interest is
preferably maintained as Agrobacterium glycerol stocks, frozen at
about -80.degree. to -90.degree. C., preferably about -86.degree.
C. The use of this preferred Agrobacterium has been found to
enhance the frequency of transformation of immature corn embryos.
As used in this invention the term "Agrobacterium capable of
transferring at least one gene" refers to Agrobacterium containing
the gene of interest, generally in a plasmid that is suitable for
mediating the events required to transfer the gene to the cells to
be infected. In a preferred embodiment, a sample of Agrobacterium
is removed from the frozen glycerol stock and grown on YP medium
for 0.5 to 5 days, preferably 1-2 days prior to co-cultivation with
the embryos.
[0028] The concentration of Agrobacterium used for co-cultivation
can affect the transformation frequency as shown by Ishida et al.
(1996) and Zhao et al. (1998). For example, while Agrobacterium can
transform immature embryos of maize, very high concentrations of
Agrobacterium may also damage the immature embryos and result in a
reduced callus response Ishida et al. (1996). To optimize the
transformation protocol for a particular maize line, immature
embryos from the maize line can be incubated with various
concentrations of Agrobacterium. Using the protocols described in
Ishida et al. (1996) and Zhao et al. (1998), the level of marker
gene expression and the transformation efficiency can be assessed
for various Agrobacterium concentrations preferably within the
concentration range of about 1.times.10.sup.7 to about
1.times.10.sup.10 cfu/ml. Using these methods, and those known in
the art, concentrations of Agrobacterium in the infection and
co-cultivation step that maximize the transformation frequency for
a particular maize line can be identified without undue
experimentation.
[0029] Preferably, Agrobacterium is used for transformations in a
concentration range of about 1.times.10.sup.8 cfu/ml to about
1.times.10.sup.10 cfu/ml, more preferably within the range of about
1.5.times.10.sup.9 cfu/ml and still more preferably at about
0.5.times.10.sup.9 cfu/ml to about 1.0.times.10.sup.9 cfu/ml. Those
skilled in the art will recognize that optimum Agrobacterium
concentration ranges may vary for particular maize genotypes and
for the particular Agrobacterium strain.
[0030] The immature embryo and Agrobacterium are co-cultivated in
accordance with conventional techniques. In the preferred
embodiment of the present invention, the isolated embryos and
Agrobacterium are co-cultivated on solid medium. Preferably the
embryos are co-cultivated with the Agrobacterium for a period of 2
to 5 days, more preferably 3 days. Preferably the solid medium is
an LS medium, which contains MS salts. Other media can also be used
such as ones which include the major inorganic salts and vitamins
of MS or N6 medium, and others well known in the art. The
co-cultivation may be performed at about 15.degree. C. to about
28.degree. C., preferably about 18.degree. C. to about 25.degree.
C., more preferably at about 19.degree. C. to 20.degree. C., and
most preferably at about 19.degree. C. It has been found that
co-cultivation at about 19.degree. C. enhances the frequency of
transformation.
[0031] In a preferred embodiment of the invention, the immature
embryo and Agrobacterium are subjected to a heat shock treatment
during co-cultivation. It has been found that this heat shock
treatment also enhances the frequency of transformation of corn
inbreds. The heat shock treatment is applied to the immature embryo
and Agrobacterium after they have been co-cultivated for about 24
to about 72 hours, preferably for about 48 to about 54 hours. It
has been found that the preferred time provides the most consistent
and reproducible results. The temperature of the heat shock is from
about 35.degree. C. to about 50.degree. C., and more preferably
about 45.degree. C. The heat shock is applied for about 20 minutes
to about 90 minutes, more preferably for about 30 minutes to about
60 minutes, and most preferably for about 30 minutes.
[0032] Any one or any combination of the three techniques described
above, may be used in accordance with the present invention to
enhance the frequency of transformation of elite corn inbreds. In
the preferred embodiment, all three techniques are used during the
co-cultivation of the immature embryo and the Agrobacterium.
[0033] Following the co-cultivation step, the "infected" embryos
are cultured to initiate the generation of Type II callus and to
grow Type II callus. It is preferred to use solid medium for the
initiation of callus tissue from the infected embryos. The solid
medium may contain any conventional salt and vitamin mixture, such
as MS salts with or without MS vitamins or other vitamins, N6 salts
with or without N6 vitamins or other vitamins and the like. The
solid medium also contains at least one antibiotic known to inhibit
the growth of Agrobacterium. In this context, it is preferred to
use cefotaxime as the antibiotic. It has been found that the
frequency of initiation of Type II callus can be enhanced by using
a staged exposure to the antibiotic. That is, the frequency of
initiation of Type II callus is enhanced by exposing the infected
embryos first to a low concentration of antibiotic and then a high
concentration of antibiotic. When cefotaxime is the antibiotic, the
low concentration is in the range of about 20 to about 100 mg/L,
preferably about 30 to about 70 mg/l, more preferably about 50 mg/L
and the high concentration is in the range of about 150 to about
300 mg/L, more preferably about 250 mg/L. The amount of other
conventionally used antibiotics can readily be determined as
described herein for cefotaxime.
[0034] It has further been found that the frequency of the
initiation of Type II callus from the infected embryos is enhanced
by including a monosaccharide in the solid medium. The
monosaccharide is in addition to the sucrose which is
conventionally present in callus initiation media. The optimum
monosaccharide for a particular elite line may be determined as
described herein. It is preferred that the monosaccharide is
glucose, maltose, lactose, sorbitol or mannitol. It is more
preferred to use glucose as the monosaccharide. The amount of
monosaccharide which is included is about 5 g/L to about 20 g/L,
more preferably about 10 g/L.
[0035] Any one or both of the two techniques described above, may
be used in accordance with the present invention to enhance the
frequency of the initiation of Type II callus during the
transformation of elite corn inbreds. In the preferred embodiment,
both techniques are used for callus initiation.
[0036] During the initiation and growth of Type II callus,
selective pressure is applied to select for those cells that have
received and are expressing polypeptide from the heterologous
nucleic acid introduced by Agrobacterium. A selective agent is
added to the solid medium on which the infected embryos are being
cultured. The agent used to select for transformants will select
for preferential growth of explants containing at least one
selectable marker insert positioned within the superbinary vector
and delivered by the Agrobacterium. For example, if the marker is
the bar gene, it confers herbicide resistance to glufosinate-type
herbicides, such as phosphinothricin (PPT) or bialaphos, and the
like. Bialaphos can then be used to select for embryos that
received and express the bar gene. Examples of other selective
markers that could be used in the vector constructs include, but
are not limited to, the pat gene, also for bialaphos and
phosphinothricin resistance, the ALS gene for imidazolinone
resistance, the HPH or HYG gene for hygromycin resistance, the EPSP
synthase gene for glyphosate resistance, the Hm1 gene for
resistance to the Hc-toxin, and other selective agents used
routinely and known to one of ordinary skill in the art. In the
preferred embodiment, the infected embryos are initially cultured
on a solid medium which does not contain a selective agent and then
transferred to medium containing a selective agent.
[0037] The initiation and growth of Type II callus free of
Agrobacterium is obtained by the use of an antibiotic and a
selective agent as described above. In accordance with a preferred
embodiment of the present invention, the infected embryos are first
cultured on a solid medium containing a monosaccharide, a low
concentration of antibiotic and no selective agent. The culturing
on this medium is performed for about 3 days to about 6 days, more
preferably for about 5 days. The embryos are then transferred to a
solid medium lacking glucose, containing a low concentration of
antibiotic and containing a selective agent. The embryos are
cultured on this medium for about 10 days to about 20 days, and
more preferably for about 14 days. Adequate control of
Agrobacterium is obtained using this protocol. It has been found
that better control of Agrobacterium growth is then obtained by
culturing the responding embryos, i.e., those embryos from which
callus tissue is developing, on a solid medium containing a high
concentration of antibiotic and a selective agent. This culturing
is performed for about 10 days to about 20 days, and more
preferably for about 14 days. Although not necessary, it is
advantageous to then culture the responding embryos on a solid
medium containing a low concentration of antibiotic and a selective
agent for about 10 days to about 20 days, and more preferably for
about 14 days. This latter culturing, if performed, is useful to
identify clones and to clear up any residual Agrobacterium growth.
The clones from either of the latter two culturings are then
cultured on a solid medium containing a selective agent and no
antibiotic to further grow and select the Type II callus prior to
plantlet regeneration. This culturing is performed for about 10
days to about 20 days, and more preferably for about 14 days.
Additional transfers on this medium may be performed as desired to
achieve further growth of clonal tissue having actively growing
Type II callus.
[0038] Actively growing Type II callus is selected from the clonal
tissue with the objective to obtain a high frequency of "water
tower" embryo structures (see FIG. 2) in the cultures. The tissue
containing the "water tower" embryo structures is cultured on a
solid medium to mature the embryos. Maturing embryos are
transferred to solid medium to further the maturation and to induce
germination. Germinating embryos are transferred to solid medium
for the promotion of further root and shoot development prior to
final transfer to soil. The solid medium may contain any
conventional salt and vitamin mixture, such as MS salts with or
without MS vitamins or other vitamins, N6 salts with or without N6
vitamins or other vitamins and the like. Methods for plant
regeneration are known in the art and preferred methods are
provided by Kamo et al. (1985), West et al. (1993), and Duncan et
al. (1985).
[0039] As discussed above with respect to the transformation method
of the present invention, it has been discovered that the
incorporation of a monosaccharide in the culturing medium for the
induction of Type II callus results in an enhanced frequency of
embryos responding. It has also been discovered that this same
effect is seen when immature embryos are cultured without
transformation. Thus, a further aspect of the present invention is
the addition of a monosaccharide to the culture medium for
initiating Type II callus. This effect is seen with any medium,
including media containing MS salts and/or vitamins, N6 salts and/
or vitamins and other conventional media. As described above, the
monosaccharide is used in an amount of about 5 g/L to about 30 g/L,
preferably about 10 g/L to about 20 g/l, more preferably about 10
g/L. The preferred monosaccharide is glucose, although other
monosaccharides can be used as shown herein.
EXAMPLES
[0040] The present invention is described by reference to the
following Examples, which are offered by way of illustration and
are not intended to limit the invention in any manner. Standard
techniques well known in the art or the techniques specifically
described below were utilized.
Example 1
Improvement in the Induction Frequency of Type II Callus
[0041] Plants of the elite corn inbred Stine 963 were grown under
controlled conditions, either in a grow room or in a greenhouse.
Plants were exposed throughout growth to a 16 h photoperiod with a
daytime temperature of around 30.degree. C. and a nighttime
temperature of around 25.degree. C. Plants were selfed and after
approximately 10 days ears were harvested. At this time the
immature embryos were on average between 1.0 and 2.0 mm in length.
Usually the ears were then placed at 4.degree. for two days prior
to immature embryo excision.
[0042] The response on standard N6A media (Table 1) for inducing
Type II callus was variable and appeared to be dependent on the
physiological state of the donor plant. The frequency of Type II
callus ranged from 0% up to 95%, with an average of around 40%.
Immature embryos were excised with a view to using them as targets
for Agrobacterium-mediated DNA delivery. For this work a
reproducible and high frequency response is required and
improvement in these aspects was therefore desirable.
[0043] The protocol used for introduction of DNA mediated by
Agrobacterium was based on that described by Hei and Komari (1997).
The co-cultivation medium in this protocol (LSAS--Table 1) contains
glucose, intended presumably as a carbon source for Agrobacterium.
After close observation of immature embryos on LSAS medium it was
thought worthwhile to check for the effects of glucose on culture
initiation and subsequent development. 10 g/l of glucose was
therefore added to N6A medium. This medium was subsequently
referred to as N6AMOD (Table 1). Results of a comparison of Type II
culture initiation on N6A and N6AMOD are shown in Table 2.
Surprisingly, N6AMOD proved to be greatly superior to N6A in
effecting Type II culture initiation from immature embryos of Stine
inbred 963.
1TABLE 1 Media Compositions Ingredients/L N6A N6AMOD LSAS MS
salts.sup.1 4.43 g N6 salts.sup.2 3.98 g 3.98 g N6 vitamins.sup.3 1
ml 1 ml Na.sub.2EDTA 10 ml Proline 700 mg 700 mg 700 mg Asparagine
150 mg 150 mg Myo-inositol 100 mg 100 mg 2,4-D 1 mg 1 mg 1.5 mg MES
500 mg 500 mg 500 mg Sucrose 20 g 20 g 20 g Glucose 10 g 10 g
Gelrite 2 g 2 g Phytagar 7 g Acetosyringone 100 .mu.m Silver
nitrate 10 mg 10 mg pH 6.0 6.0 5.8 .sup.1MS salts -- Sigma Plant
Culture Catalogue ref M 5519 .sup.2N6 salts -- Sigma Plant Culture
Catalogue ref. C 1416 .sup.3N6 vitamins: 2 mg/L glycine, 0.5 mg/L
nicotinic acid, 0.5 mg/L pyridoxine-HCl, 1 mg/L thiamine-HCl (Chu,
1978).
[0044]
2TABLE 2 Effect of Glucose on Frequency of Embryos Responding No.
of Immature No. of Immature % Medium Embryos Cultured Embryos
Responding Responding N6A 1034 466 45.1 N6AMOD 965 751 77.8
[0045] Various other sugars were tested in this regard (see Table
3) and some are capable of supporting an increased rate of Type II
callus initiation (especially maltose, lactose, sorbitol and
mannitol). None of those tested, however, were as efficient as
glucose in stimulating Type II callus formation on N6A medium. All
sugars were tested at 10 g/l in combination with 20 g/l
sucrose.
3TABLE 3 Effects of Different Sugars on Frequency of Embryos
Responding Sugar Embryo Response Glucose >75% Maltose 50%-60%
Lactose 50%-60% Sorbitol 50%-60% Mannitol 50%-60% Raffinose <50%
Mellibiose <50% Cellobiose <50% Fructose <50% Xylose
<50% Trehalose <50% Galactose <30% Control 46%
[0046] Similar results were achieved when glucose or other
monosaccharide (which demonstrated an enhanced frequency of callus
initiation in this example) is included in callus initiation media
of the prior art containing other mineral salts and vitamins. Thus,
the use of a monosaccharide in the callus induction medium enhances
the frequency of callus induction.
Example 2
Transformation of an Elite Corn Inbred by Agrobacterium
tumefaciens
[0047] Modifications to the protocol of Hei and Komari (1997)
involving co-cultivation temperature, culture media, antibiotic
concentrations and Agrobacterium source cultures.
[0048] Agrobacterium strain LBA 4404 harboring "super binary"
vectors as described in U.S. patent Hei and Komari (1997) was used
in corn transformation experiments. Vectors with a bar expression
cassette from pBARGUS (Fromm et. al., 1990) were used to generate
resistance to the herbicide bialaphos, and a gus expression
cassette from pIG221 (Ohta et al., 1990) was used to produce Gus
expression for transient assays as displayed in FIG. 1. The gus
expression cassette contains an intron in the N-terminal region of
the gus gene which prevents expression in bacteria, but upon
expression in plant cells the intron is spliced out and Gus
activity is achieved (Ohta et al., 1990; Ishida et al., 1996).
Agrobacterium containing "super binary" vectors were stored in
glycerol stocks using acidified glycerol. Glycerol was acidified by
adding 15 drops of 1M HCl to one liter of glycerol (Sigma G-9012).
Final glycerol concentration of stocks was 15 to 20% and stocks
were frozen at minus 86.degree. C. When glycerol stocks were used
as the source for transformation experiments, Agrobacterium was
made ready for transformation experiments by removing a few flakes
of frozen culture with a sterile loop, streaking it out on YP
medium (5 g/l yeast extract, 10 g/l peptone, 5 g/l NaCl, and 15 g/l
agar) containing 50 mg/l spectinomycin, and incubating it for one
or two days at 28.degree. C. When glycerol stocks were not used as
the source, Agrobacterium maintained on YP plus spectinomycin at
4.degree. C. was used to initiate new cultures of Agrobacterium
that were grown as described above.
[0049] Co-cultivation of the immature embryos and Agrobacterium
cells in plant transformation work has been routinely performed at
25.degree. C. Observations by Fullner et al. (1996) suggested that
better results might be expected at lower temperatures. This was
confirmed by Dillen et al. (1997) for transformation of tobacco. We
therefore tested 19.degree. C. as a co-cultivation temperature for
corn. FIG. 1 shows the clear superiority of co-cultivating at
19.degree. C. as indicated by transient expression of the gus gene.
Subsequently, all experiments were carried out at a co-cultivation
temperature of 19.degree. C. The protocol of Hei and Komari (1997)
utilizes the corn inbred line A188 and hybrids with A188. No
success was reported with other inbreds (Ishida et al., 1996).
Their approach was tried with Stine 963 and was not successful.
Cultured immature embryos of Stine 963 treated with Agrobacterium
after Hei and Komari, and Ishida et al produced no transformed
clones. The following modifications were then tried:
[0050] (a) Stine 963 embryos, after co-cultivation on LSAS for
three days, were transferred to N6A medium for production of Type
II callus for subsequent selection (rather than LS 1.5D as
described by Ishida et al., 1996). This allowed for at least some
embryo response. 1901 embryos were co-cultivated on LSAS and then
cultured and selected on N6A-based media. 3 clones were recovered
from this approach (0.15%).
[0051] (b) It was noted that Agrobacterium growth was inhibited on
media containing silver nitrate. It was also noted that 250 mg/l
cefotaxime (concentration of the antibiotic used by Ishida et al to
control Agrobacterium growth) severely inhibited embryo response.
Therefore, a lower level of cefotaxime was tested in the presence
of silver nitrate to see if embryo response and subsequent clone
recovery could be improved. Adequate control of Agrobacterium
growth for the first 19 day culture period after co-cultivation was
obtained with 50 mg/l cefotaxime (DN62ALC (5 days) and DN62ALCB (14
days)--Table 4) but not with 10 mg/l. Further experiments indicated
that it was advantageous to culture responding immature embryos on
250 mg/l cefotaxime (DN62ACB--Table 4) for 14 days after the first
14 day passage on DN62ALCB for better control of Agrobacterium
growth. Finally, a further 14 day passage on DN62ALCB was required
to identify clones and to clear up any residual Agrobacterium
growth. The clones were then transferred to DN62B (Table 4) for
growth and further selection prior to regeneration. Using this
scheme less than 1% of clones identified and cultured showed any
evidence of residual Agrobacterium growth.
[0052] The media described above (DN62ALC, DN62ALCB and DN62ACB)
combine the improved survival of immature embryos with staged
exposure to cefotaxime, with the improved reproducibility and high
frequency of response obtained with initial exposure to glucose (on
DN62ALC). With these modifications 2167 embryos were co-cultivated
on LSAS and then cultured and selected on DN62ALC, DN62ALCB,
DN62ACB and finally DN62ALCB. 18 clones were recovered (0.83%).
4TABLE 4 Media Compositions Ingredients/L DN62B DN62ALC DN62ALCB
DN62ACB N6 salts.sup.1 3.98 g 3.98 g 3.98 g 3.98 g N6
vitamins.sup.1 1 ml 1 ml 1 ml 1 ml Asparagine 800 mg 800 mg 800 mg
800 mg Myo-inositol 100 mg 100 mg 100 mg 100 mg Proline 1400 mg
1400 mg 1400 mg 1400 mg Casamino Acids 100 mg 100 mg 100 mg 100 mg
2,4-D 1 mg 1 mg 1 mg 1 mg Sucrose 20 g 20 g 20 g 20 g Glucose 10 g
AgNO.sub.3 10 mg 10 mg 10 mg Bialaphos 1 mg 1 mg 1 mg Cefotaxime 50
mg 50 mg 250 mg Gelrite 3 g 3 g 3 g 3 g pH 5.8 5.8 5.8 5.8 .sup.1N6
salts and vitamins: See Table 1.
[0053] (c) Certain individual experiments were noted as having
produced a relatively large number of transformed clones. On
further analysis, one common factor was determined to be use of
Agrobacterium source cultures recently recovered from frozen
glycerol stocks maintained at -86.degree. C. A comparison was then
made between Agrobacterium cells taken after one or two days
culture on YP medium immediately after recovery from glycerol
stocks, with Agrobacterium cells maintained on YP medium for a
month or longer after recovery from glycerol stocks. The surprising
result is noted in Table 5.
5 TABLE 5 No. of Immature Number % Embryos Co-Cultivated of Clones
Response YP Stocks.sup.1 1530 25 1.6 Glycerol Stocks.sup.2 933 58
6.2 .sup.1Combined data from 10 separate experiments.
.sup.2Combined data from 9 separate experiments.
Example 3
Effect of Heat Shock Treatment on Integration of DNA
[0054] Use of a brief heat treatment induces a transient state of
so-called `competence` in bacteria, allowing them to take up and
express DNA from a variety of sources (cited in Sambrook et al,
1989). Use of a heat shock treatment to improve transformation
efficiencies in higher organisms has not been reported. It was
decided to explore the possibility that a heat shock treatment
could improve integration of DNA following uptake. This was
investigated with Agrobacterium-mediated DNA delivery in the first
instance. First, a heat shock treatment of 45.degree. for 30
minutes was administered to immature embryos 21, 24, 27, and 30
hours after the initiation of co-cultivation with Agrobacterium on
LSAS (Table 1). No enhancement of clone production was noted after
21 hours, but promising preliminary results were obtained with the
longer time periods. Further experiments were then performed--the
results are presented in Table 6.
6TABLE 6 Effect of Heat Shock on Clone Recovery Heat Shock No. of
Embryos No. of Clones % Treatment.sup.1 Co-Cultivated Recovered
Response (A) 45.degree./30 min/24 h 104 46 44.2 Control 225 11 4.9
(B) 45.degree./30 min/48-54 h 208 120 57.6 Control 268 13 4.8
.sup.1Temperature and duration of heat shock at specified time
after initiation of co-cultivation.
[0055] From Table 6 it can be seen that an approximately ten-fold
improvement in the frequency of clone production was obtained
following heat shock treatment. Although high frequencies of
response could be obtained in some experiments following a heat
shock treatment administered after 24 hours, more consistent and
reproducible results were obtained when the heat shock was
administered after 48 to 54 hours. Enhancement in frequency of
clone production was also noted when the heat shock was
administered for 60 minutes instead of 30.
Example 4
Regeneration of Plants
[0056] Clones could be induced to regenerate plants by the
following procedures and media manipulations. Presence of the bar
gene was confirmed by leaf painting with Liberty, both in the
primary transformants and in progeny where Mendelian ratios were
routinely observed.
[0057] (a) Actively growing Type II callus was selected from clonal
tissue, with the objective of obtaining a high frequency of
so-called `water tower` embryo structures (see FIG. 2) in the
cultures.
[0058] (b) These tissues were then transferred to DNROB medium
(Table 7). On this medium embryo maturation occurred.
[0059] (c) Maturing tissues were then transferred off DNROB after
two or three weeks either to a fresh plate of DNROB or to O-INABAGS
(Table 7). After a further one to two weeks, embryos with a shoot
meristem were placed on MSOG medium or 1/2MSIBA (Table 7), where
germination occurred. Plantlets were then transferred to tubes
containing 1/2MSIBA medium for promotion of further root and shoot
development prior to final transfer to soil.
7TABLE 7 Media Compositions Ingredients/L DNROB O-INABAGS MSOG
1/2MSIBA MS Salts.sup.1 4.43 g 4.43 g 4.43 g 2.215 g Asparagine 800
mg Proline 1400 mg Na.sub.2EDTA 37.3 mg 37.3 mg 37.3 mg 37.3 mg
Casamino Acids 100 mg Nicotinic Acid 0.5 mg Gibberellic Acid 0.1 mg
NAA 0.1 mg Indole-3-Butyric 0.1 mg Acid ABA 0.13 mg Sucrose 60 g 30
g 20 g Sorbitol 20 g Bialaphos 1 mg Gelrite 2 g Phytagar 7 g 7 g 7
g pH 5.8 5.8 5.8 5.8 .sup.1MS Salts -- Sigma Plant culture
Catalogue ref M5519.
[0060] It will be appreciated that the methods and compositions of
the instant invention can be incorporated in the form of a variety
of embodiments, only a few of which are disclosed herein. It will
be apparent to the artisan that other embodiments exist and do not
depart from the spirit of the invention. Thus, the described
embodiments are illustrative and should not be construed as
restrictive.
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* * * * *